Two-photon microscopes and multiphoton tomographs (MPT) in which fluorescence signals and SHG (Second Harmonic Generation) signals are excited in biological molecules in vivo by pulsed laser radiation in the near infrared region and are detected by suitable highly sensitive, fast receivers are known (e.g., U.S. Pat. No. 5,034,613 A, DE 201 17 294 U1; DE 203 06 122 U1). Further, nonfluorescing and non-SHG active components such as water and lipids can be displayed by means of CARS (Coherent Anti-Stokes Raman Scattering) microscopes and CARS tomographs (see, e.g., DE 10 2010 047 578 A1, not previously published).
Horizontal resolutions in the submicrometer range and scan areas (object regions) of several hundred micrometers in three dimensions are achieved by these nonlinear optical microscopes and tomographs.
Nonlinear microscopes and tomographs of this kind are based on the use of rigid designs and vibration-damped optical stage arrangements. They can be operated as either upright or inverted optical microscope arrangements. However, no high-resolution imaging systems (with a lateral resolution of about 1 μm or less) permitting a free positioning of the measuring head at the required lateral and axial resolution in the submicrometer range are known for nonlinear microscopy or tomography.
A beam transmission required for this purpose can be realized by optical fiber systems or as a free-beam transmission system. The accuracy of free-beam laser transmission systems such as are known, for example, in the form of articulated mirror arms is affected by mechanical deviations, mechanical stresses and temperature drift within the mechanical cage structure of these arms. In various configurations of the articulated arm, the effect on the mechanical axis of the articulated mirror arm (due to temperature drift, for example) leads to a deviation of the position of the laser beam at the output of the articulated arm relative to the target position or mechanical axis so that a reliable scanning of a measurement object by means of high-resolution focusing optics at different orientations of the measuring head is possible only to a limited extent. The fluctuations in the position of the laser beam at the output of the optical articulated arm can be subsumed under the concept of “accuracy of the transmission system.”
The accuracy of the laser beam transmission is further influenced by the given length of the optical beam path of an articulated arm and the beam position stability (pointing ability) of the laser.
In the present instance, pointing ability refers to the change in the directional stability of the laser radiation at the direct output of the laser resulting from the thermally induced effect on the resonator configuration of the laser.
The coupling of the laser radiation into an articulated mirror arm is especially critical. In-coupling is optimal when the optical axis of the laser beam coincides with the mechanical (entrance) axis of the articulated arm (collinearity). Deviations occurring in this respect influence accuracy apart from the above-mentioned mechanically caused and thermally caused deviations of the articulated arm.
While the deviations to be compensated in guiding the laser beam are reduced exclusively to the in-coupling and out-coupling of the beam when using fiber transmission optics, the basic problem of undesirable laser beam deviation at the target site remains. In particular, there is also the risk of unwanted coupling into the fiber sheath (cladding).
U.S. Pat. No. 5,463,215 A discloses a device for aligning a light beam for coupling thereof into an optical fiber in which a portion of the laser beam that is not coupled into the fiber diameter is coupled out to a detector device via two annular conical mirrors which are arranged as retroreflectors and a mirror rotating on a ring at 45°. By synchronizing the detector with the position of the revolving mirror, a deviation from the collinearity of the laser axis and fiber axis and a defocusing can be detected. Further, an overlapping illumination of the fiber input (i.e., beam diameter greater than fiber diameter) is required and, therefore, a loss of intensity is already inevitable from the outset.
Accordingly, the prior art presents the following disadvantages:                owing to their rigid construction, conventional two-photon microscopes and multiphoton tomographs (MPTs) present a limited range of application and are, for example, unsuitable for multifunctional examinations on the human body;        for realizing a flexible construction, the mechanical tolerances of beam-guiding systems are too large to ensure a sufficiently constant illumination of a measurement spot for a reproducible irradiation of the scan site permitting a submicrometer resolution; and        in multiphoton microscopy, the limitation of imaging to the detection of autofluorescence of endogenous substances and to nonlinear frequency multiplication of certain molecules is often disadvantageous, and the desirable combination with CARS systems could only be realized heretofore in rigid systems because of the strict requirements respecting collinearity.        